The origin, lineage derivation, and sites of human natural killer (NK) cell differentiation are presently unresolved. The vast majority of NK cells found in peripheral blood have surface membrane expression of CD2 and CD16. Both antigens trigger activation pathways which require the zeta protein, a signal-transducing subunit of the CD3-T cell receptor (CD3-TCR) complex which is found as an isolated homodimer (zeta-zeta) or heterodimer (zeta-Fc epsilon RI gamma) in human NK cells. Unlike NK cells found in adult peripheral blood, NK cells derived in vitro from human CD34+ hematopoietic progenitor cells lack CD2 and CD16, and those found in fetal liver constitutively express CD3 epsilon and delta proteins. However, NK effectors derived in vitro from immature human CD3- thymocytes show striking phenotypic and functional similarities to adult human NK cells. In this report, we characterize zeta protein expression in CD3- thymocytes following short-term culture in recombinant (r)IL-2. CD3-CD56+ thymocyte NK effectors express the zeta protein as a disulphide-linked homodimer of 32 kDa, yet lack other protein components of the CD3-TCR complex. Both CD16+ and CD16- populations were found to express zeta, and within the CD16+ fraction, zeta is physically associated with CD16. These data provide evidence of additional similarities between adult peripheral blood NK cells and CD3-CD56+ NK effectors derived from human thymocytes, and suggest that under these experimental conditions, human NK cells can arise from early thymic precursors.